U.S. patent number 11,078,534 [Application Number 16/199,757] was granted by the patent office on 2021-08-03 for photocleavable nucleotide reagents with high stability.
This patent grant is currently assigned to Roche Molecular Systems, Inc.. The grantee listed for this patent is Roche Molecular Systems, Inc.. Invention is credited to Concordio Anacleto, Amar Gupta, Dieter Heindl, Igor Kozlov, Hannes Kuchelmeister, Alexander Nierth, Christian Wellner, Stephen Gordon Will.
United States Patent |
11,078,534 |
Nierth , et al. |
August 3, 2021 |
Photocleavable nucleotide reagents with high stability
Abstract
The present invention provides for stable nucleotide reagents
used for nucleic acid amplification by PCR and RT-PCR (Reverse
Transcriptase-PCR) that comprises modified nucleoside
triphosphates. The present invention also provides for methods for
using the modified nucleoside triphosphates for detecting the
presence or absence of a target nucleic acid sequence in a sample
in an amplification reaction.
Inventors: |
Nierth; Alexander (Dublin,
CA), Kuchelmeister; Hannes (Munich, DE), Anacleto;
Concordio (Dublin, CA), Gupta; Amar (Danville, CA),
Heindl; Dieter (Munich, DE), Kozlov; Igor
(Danville, CA), Wellner; Christian (Penzberg, DE),
Will; Stephen Gordon (Oakland, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Molecular Systems, Inc. |
Pleasanton |
CA |
US |
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Assignee: |
Roche Molecular Systems, Inc.
(Pleasanton, CA)
|
Family
ID: |
1000005716921 |
Appl.
No.: |
16/199,757 |
Filed: |
November 26, 2018 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20190161797 A1 |
May 30, 2019 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62590972 |
Nov 27, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H
19/10 (20130101); C12Q 1/686 (20130101); C07H
19/20 (20130101); C12Q 1/6848 (20130101); C12Q
1/6876 (20130101); C07H 21/02 (20130101); C12Q
1/6848 (20130101); C12Q 2523/319 (20130101); C12Q
1/686 (20130101); C12Q 2523/319 (20130101) |
Current International
Class: |
C12Q
1/68 (20180101); C12Q 1/6848 (20180101); C12Q
1/686 (20180101); C07H 19/20 (20060101); C07H
21/02 (20060101); C07H 19/10 (20060101); C12Q
1/6876 (20180101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Stratagene Catalog p. 39. (Year: 1988). cited by examiner .
International Search Report and Written Opinion dated Jan. 23, 2019
in Application No. PCT/EP2018/082692, 10 pages. cited by applicant
.
Tang, X., et al., Caged nucleotides/nucleosides and their
photochemical biology, Organic & Biomolecular Chemistry, Jan.
1, 2013, pp. 7814-7824, vol. 11, No. 45. cited by applicant .
Kretschy, N. et al., "Next-Generation o-Nitrobenzyl Photolabile
Groups for Light-Directed Chemistry and Microarray Synthesis",
Angew. Chem. Int. Ed., 2015, 54, 8555-8559. cited by
applicant.
|
Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Chang; David J.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority of U.S. Provisional
Patent Application Ser. No. 62/590,972, filed Nov. 27, 2017, which
is incorporated herein by reference in its entirety.
Claims
The invention claimed is:
1. A method of detecting the presence or absence of a target
nucleic acid sequence in a sample comprising: a) performing an
amplifying step comprising contacting the sample with amplification
reagents to produce an amplification product if the target nucleic
acid sequence is present in the sample, and b) detecting the
amplification product, wherein the amplification reagents comprise
a modified nucleoside triphosphate having a photocleavable moiety
and having a structure of: ##STR00007## where Base is a purine or
pyrimidine base or an analog; and wherein prior to step a), the
modified nucleoside triphosphate is irradiated with light having a
wavelength capable of cleaving the photocleavable moiety.
2. A method of amplifying a target nucleic acid sequence in a
sample comprising: providing amplification reagents to perform an
amplifying step, wherein the amplification reagents comprise a
modified nucleoside triphosphate having a photocleavable moiety and
having a structure of: ##STR00008## where Base is a purine or
pyrimidine base or an analog; and wherein prior to the amplifying
step, the modified nucleoside triphosphate is irradiated with light
having a wavelength capable of cleaving the photocleavable
moiety.
3. A method of reducing or preventing non-specific amplification of
nucleic acid in a sample during amplification by polymerase chain
reaction (PCR) comprising: a) providing amplification reagents that
are used to amplify a target nucleic acid, wherein the
amplification reagents comprise a nucleic acid polymerase and a
modified nucleoside triphosphate having a photocleavable moiety and
having a structure of: ##STR00009## where Base is a purine or
pyrimidine base or an analog; b) contacting the sample with the
amplification reagents at room temperature, whereby the
photocleavable moiety on the modified nucleoside triphosphate
prevents processing by the nucleic acid polymerase, thereby
suppressing formation of non-specific amplification products from
room temperature activity of the nucleic acid polymerase; c) prior
to amplifying, irradiating the modified nucleoside triphosphate
with light having a wavelength capable of cleaving the
photocleavable moiety to release free nucleoside triphosphate; and
d) amplifying the target nucleic acid by PCR.
4. A modified nucleoside triphosphate having a photocleavable
moiety and having a structure of: ##STR00010## where Base is a
purine or pyrimidine base or an analog.
5. The modified nucleoside triphosphate of claim 4 characterized by
having greater thermostability compared to an unmodified nucleoside
triphosphate.
6. A kit comprising the modified nucleoside triphosphate of claim 4
and further comprising reagents necessary for performing an
amplification reaction.
7. A kit comprising the modified nucleoside triphosphate of claim 5
and further comprising reagents necessary for performing an
amplification reaction.
Description
FIELD OF THE INVENTION
The present invention provides for stable nucleotide reagents,
methods for their preparation, methods for their use, and kits
comprising them. The nucleotide reagents are useful in many
recombinant DNA techniques, especially nucleic acid amplification
by the polymerase chain reaction (PCR).
BACKGROUND OF THE INVENTION
Nucleic acid amplification reagents are typically comprised of
temperature sensitive components, and therefore must often be
stored and shipped at temperatures well below ambient temperature.
This is particularly the case with deoxynucleoside triphosphates or
their ribonucleoside triphosphate analogs. These reagents are prone
to degradation via loss of consecutive phosphate groups from the
termini, resulting in the formation of nucleoside diphosphates and
monophosphates, both of which are no longer active as substrates of
nucleic acid polymerases. The stability of nucleoside
polyphosphates can be improved substantially by esterifying the
terminal phosphates. For example, .gamma.-methyl-dNTP analogs were
completely stable under conditions of heat stress that were
sufficient to completely degrade normal dNTPs. However,
esterification of the terminal phosphate can have a negative effect
on the ability of these nucleotides to serve as effective
substrates for certain polymerase enzymes. A need exists for having
nucleoside triphsosphates that are both thermally stable and are
inactive initially as substrates but are readily activated by
simple steps.
BRIEF SUMMARY OF THE INVENTION
The present invention provides for thermally stable nucleotide
reagents used for nucleic acid amplification by PCR and RT-PCR
(Reverse Transcriptase-PCR) that comprises modified nucleoside
polyphosphates having a photocleavable moiety. The present
invention also provides for methods for using the modified
nucleoside triphosphates for detecting the presence or absence of a
target nucleic acid sequence in a sample in an amplification
reaction.
Therefore in one aspect, the present invention involves a method of
detecting the presence or absence of a target nucleic acid sequence
in a sample comprising performing an amplifying step comprising
contacting the sample with amplification reagents to produce an
amplification product if the target nucleic acid sequence is
present in the sample, and detecting the amplification product,
wherein the amplification reagents comprise a modified nucleoside
triphosphate having a photocleavable moiety and having a structure
of:
##STR00001## where Base is a purine or pyrimidine base, or an
analog. In one embodiment, prior to the amplifying step, the
modified nucleoside polyphosphate is irradiated with light having a
wavelength capable of cleaving the photocleavable moiety.
In another aspect, the present invention involves a method of
amplifying a target nucleic acid sequence using amplification
reagents wherein the amplification reagents comprise a modified
nucleoside triphosphate having photocleavable moiety and having a
structure of:
##STR00002## where Base is a purine or pyrimidine base, or an
analog. In one embodiment, prior to amplifying, the modified
nucleoside triphosphate is irradiated with light having a
wavelength capable of cleaving the photocleavable moiety.
In another aspect, the present invention involves a method of
reducing or preventing non-specific amplification of nucleic acid
during an amplification reaction comprising providing amplification
reagents that are used to amplify a target nucleic acid, wherein
the amplification reagents comprise a modified nucleoside
triphosphate having a photocleavable moiety and having a structure
of:
##STR00003## where Base is a purine or pyrimidine base or an
analog. In one embodiment, prior to amplifying, the modified
nucleoside triphosphate is irradiated with light having a
wavelength capable of cleaving the photocleavable moiety.
In another aspect, the present invention involves a composition
comprising a modified nucleoside triphosphate having photocleavable
moiety and having a structure of:
##STR00004## where Base is a purine or pyrimidine base, or an
analog. In one embodiment, the modified nucleoside triphosphate is
characterized by having greater thermostability compared to an
unmodified nucleoside triphosphate.
In yet another aspect, the present invention provides for a kit
comprising the modified nucleoside triphosphate and further
comprising reagents necessary for performing an amplification
reaction.
The embodiments and advantages of the invention are described in
more detail in the Detailed Description of the Invention and in the
Figures.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the structure and general concept of the
photocleavable nucleoside triphosphate, thiophenyl
o-nitrophenylpropyl dNTP.
FIG. 2 shows the molecular structures of known photocleavable
(d)NTPs.
FIG. 3 is a schematic for the prepration of thiphenyl
o-nitrophenylpropyl dATP (dATP-SPh-NPP).
FIG. 4 shows the thermostability of thiophenyl o-nitrophenylpropyl
dATP (dATP-SPh-NPP) compared to regular dATP. The samples were
exposed to 65.degree. C. for up to 120 h.
FIG. 5 shows the photocleavage of thiophenyl o-nitrophenylpropyl
dATP (dATP-SPh-NPP) compared to DMNPE-ATP, a commercially available
photocleavable nucleotide, with LED UV flashlight in 100 mM
phosphate buffer, pH 7.0 at room temperature.
FIG. 6 shows the results of PCR amplification reactions performed
with regular dGTP, dCTP, dUTP and caged dATP-SPh-NPP without
(graphs on left column) and with (graphs on right column) exposure
to UV light for 8 minutes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for thermally stable modified
nucleoside triphosphate having a photocleavable moiety, methods for
their preparation, methods for their use, and kits comprising them.
These thermally stable modified nucleoside triphosphates are useful
in many recombinant DNA techniques, especially nucleic acid
amplification by the polymerase chain reaction (PCR).
Definitions
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although essentially any methods and materials similar to those
described herein can be used in the practice or testing of the
present invention, only exemplary methods and materials are
described. For purposes of the present invention, the following
terms are defined below.
The terms "a," "an," and "the" include plural referents, unless the
context clearly indicates otherwise.
The term "ambient temperature" refers to the temperature of the
surrounding and is synonymous with "room temperature" when
referring to the temperature of a temperature-controlled indoor
building. Typically, ambient temperature refers to a temperature
range of between 15.degree. C. and 25.degree. C. although slightly
cooler or warmer temperatures may still be considered within the
range of ambient temperature.
The term "photocleavable moiety" that is used herein has several
synonymous words in the literature and can also be referred to as
"photoremovable groups", "photoblocking groups", "phototriggers",
"caged compounds". "photolabile groups", and other similar terms.
One advantage of employing a photocleavable moiety is the ability
to control reactions by temporally blocking or masking its function
with high selectivity. The photocleavable moiety can be selected to
be cleaved, i.e., decoupled, from the nucleoside triphosphate with
a light of a certain wavelength, for example a wavelength of
between about 200 nm to about 450 nm, e.g., between about 200 nm to
about 300 nm, or e.g., between about 280 nm to about 315 nm, or
e.g., between about 300 nm to about 400 nm, or e.g., between about
315 nm to about 415 nm. The term "about" in the context of a stated
wavelength may include exactly the stated wavelength, and also
include a wavelength having 1 nm, 2 nm, 3 nm, 4 nm, or 5 nm, plus
or minus the stated wavelength. Thus, when a light of appropriate
wavelength is introduced, e.g., by shining or flashing, to the
modified nucleoside triphosphate, the photocleavable moiety is
cleaved which removes the steric or other interferences from
processing by nucleic acid polymerases. The introduction of the
light can be manual or automated. Once the light is introduced and
the photocleavable moieties are removed, the restored nucleoside
triphosphate can become a substrate for the polymerase during the
PCR reaction.
"Recombinant", as used herein, refers to an amino acid sequence or
a nucleotide sequence that has been intentionally modified by
recombinant methods. By the term "recombinant nucleic acid" herein
is meant a nucleic acid, originally formed in vitro, in general, by
the manipulation of a nucleic acid by restriction endonucleases, in
a form not normally found in nature. Thus an isolated, mutant DNA
polymerase nucleic acid, in a linear form, or an expression vector
formed in vitro by ligating DNA molecules that are not normally
joined, are both considered recombinant for the purposes of this
invention. It is understood that once a recombinant nucleic acid is
made and reintroduced into a host cell, it will replicate
non-recombinantly, i.e., using the in vivo cellular machinery of
the host cell rather than in vitro manipulations; however, such
nucleic acids, once produced recombinantly, although subsequently
replicated non-recombinantly, are still considered recombinant for
the purposes of the invention. A "recombinant protein" is a protein
made using recombinant techniques, i.e., through the expression of
a recombinant nucleic acid as depicted above.
A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the sequence; or a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to facilitate translation.
The term "host cell" refers to both single-cellular prokaryote and
eukaryote organisms (e.g., bacteria, yeast, and actinomycetes) and
single cells from higher order plants or animals when being grown
in cell culture.
The term "vector" refers to a piece of DNA, typically
double-stranded, which may have inserted into a piece of foreign
DNA. The vector or may be, for example, of plasmid origin. Vectors
contain "replicon" polynucleotide sequences that facilitate the
autonomous replication of the vector in a host cell. Foreign DNA is
defined as heterologous DNA, which is DNA not naturally found in
the host cell, which, for example, replicates the vector molecule,
encodes a selectable or screenable marker, or encodes a transgene.
The vector is used to transport the foreign or heterologous DNA
into a suitable host cell. Once in the host cell, the vector can
replicate independently of or coincidental with the host
chromosomal DNA, and several copies of the vector and its inserted
DNA can be generated. In addition, the vector can also contain the
necessary elements that permit transcription of the inserted DNA
into an mRNA molecule or otherwise cause replication of the
inserted DNA into multiple copies of RNA. Some expression vectors
additionally contain sequence elements adjacent to the inserted DNA
that increase the half-life of the expressed mRNA and/or allow
translation of the mRNA into a protein molecule. Many molecules of
mRNA and polypeptide encoded by the inserted DNA can thus be
rapidly synthesized.
"Amplification reagents" are chemical or biochemical components
that enable the amplification of nucleic acids. Such reagents
comprise, but are not limited to, nucleic acid polymerases,
buffers, mononucleotides such as nucleoside triphosphates,
oligonucleotides e.g. as oligonucleotide primers, salts and their
respective solutions, detection probes, dyes, and more.
As is known in the art, a "nucleoside" is a base-sugar combination.
The base portion of the nucleoside is normally a heterocyclic base.
The two most common classes of such heterocyclic bases are purines
and pyrimidines.
"Nucleotides" are nucleosides that further include a phosphate
group covalently linked to the sugar portion of the nucleoside. For
those nucleosides that include a pentofuranosyl sugar, the
phosphate group can be linked to either the 2'-, 3'- or 5'-hydroxyl
moiety of the sugar. A nucleotide is the monomeric unit of an
"oligonucleotide", which can be more generally denoted as an
"oligomeric compound", or a "polynucleotide", more generally
denoted as a "polymeric compound". Another general expression for
the aforementioned is deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA).
An "oligomeric compound" is a compound consisting of "monomeric
units" which may be nucleotides alone or non-natural compounds (see
below), more specifically modified nucleotides (or nucleotide
analogs) or non-nucleotide compounds, alone or combinations
thereof.
"Oligonucleotides" and "modified oligonucleotides" (or
"oligonucleotide analogs") are subgroups of oligomeric compounds.
The term "oligonucleotide" refers to components formed from a
plurality of nucleotides as their monomeric units. The phosphate
groups are commonly referred to as forming the internucleoside
backbone of the oligonucleotide. The normal linkage or backbone of
RNA and DNA is a 3' to 5' phosphodiester linkage. Oligonucleotides
and modified oligonucleotides (see below) useful for the invention
may be synthesized as principally described in the art and known to
the expert in the field. Methods for preparing oligomeric compounds
of specific sequences are known in the art, and include, for
example, cloning and restriction of appropriate sequences and
direct chemical synthesis. Chemical synthesis methods may include,
for example, the phosphotriester method described by Narang S. A.
et al., Methods in Enzymology 68 (1979) 90-98, the phosphodiester
method disclosed by Brown E. L., et al., Methods in Enzymology 68
(1979) 109-151, the phosphoramidite method disclosed in Beaucage et
al., Tetrahedron Letters 22 (1981) 1859, the H-phosphonate method
disclosed in Garegg et al., Chem. Ser. 25 (1985) 280-282 and the
solid support method disclosed in U.S. Pat. No. 4,458,066.
In the process described above, the oligonucleotides may be
chemically modified, i.e. the primer and/or the probe comprise of a
modified nucleotide or a non-nucleotide compound. The probe or the
primer is then a modified oligonucleotide.
"Modified nucleotides" (or "nucleotide analogs") differ from a
natural nucleotide by some modification but still consist of a
base, a pentofuranosyl sugar, a phosphate portion, base-like,
pentofuranosyl sugar-like and phosphate-like portion or
combinations thereof. For example, a label may be attached to the
base portion of a nucleotide whereby a modified nucleotide is
obtained. A natural base in a nucleotide may also be replaced by
e.g. a 7-deazapurine whereby a modified nucleotide is obtained as
well.
A "modified oligonucleotide" (or "oligonucleotide analog"),
belonging to another specific subgroup ofoligomeric compounds,
possesses one or more nucleotides and one or more modified
nucleotides as monomeric units. Thus, the term "modified
oligonucleotide" (or "oligonucleotide analog") refers to structures
that function in a manner substantially similar to oligonucleotides
and can be used interchangeably in the context of the present
invention. From a synthetical point of view, a modified
oligonucleotide (or an oligonucleotide analog) can for example be
made by chemical modification of oligonucleotides by appropriate
modification of the phosphate backbone, ribose unit or the
nucleotide bases (Uhlmann and Peyman, Chem. Rev. 90 (1990) 543;
Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134).
Representative modifications include phosphorothioate,
phosphorodithioate, methyl phosphonate, phosphotriester or
phosphoramidate inter-nucleoside linkages in place of
phosphodiester internucleoside linkages; deaza- or azapurines and
-pyrimidines in place of natural purine and pyrimidine bases,
pyrimidine bases having substituent groups at the 5 or 6 position;
purine bases having altered substituent groups at the 2, 6 or 8
positions or 7 position as 7-deazapurines; bases carrying alkyl-,
alkenyl-, alkinyl or aryl-moieties, e.g. lower alkyl groups such as
methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl,
octyl, nonyl, decyl, or aryl groups like phenyl, benzyl, naphtyl;
sugars having substituent groups at, for example, their 2'
position; or carbocyclic or acyclic sugar analogs. Other
modifications are known to those skilled in the art. Such modified
oligonucleotides (or oligonucleotide analogs) are best described as
being functionally interchangeable with, yet structurally different
from, natural oligonucleotides. In more detail, exemplary
modifications are disclosed in Verma S., and Eckstein F., Annu.
Rev. Biochem. 67 (1998) 99-134 or WO 02/12263. In addition,
modification can be made wherein nucleoside units are joined via
groups that substitute for the internucleoside phosphate or sugar
phosphate linkages. Such linkages include those disclosed in Verma
S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. When
other than phosphate linkages are utilized to link the nucleoside
units, such structures have also been described as
"oligonucleosides".
A "nucleic acid" as well as the "target nucleic acid" is a
polymeric compound of nucleotides as known to the expert skilled in
the art. "Target nucleic acid" is used herein to denote a nucleic
acid in a sample which should be analyzed. i.e. the presence,
non-presence and/or amount thereof in a sample should be
determined.
The term "primer" is used herein as known to the expert skilled in
the art and refers to oligomeric compounds, primarily to
oligonucleotides, but also to modified oligonucleotides that are
able to prime DNA synthesis by a template-dependent DNA polymerase,
i.e. the 3'-end of the e.g. primer provides a free 3'--OH group
whereto further nucleotides may be attached by a template-dependent
DNA polymerase establishing 3'- to 5'-phosphodiester linkage
whereby deoxynucleoside triphosphates are used and whereby
pyrophosphate is released.
A "probe" also denotes a natural or modified oligonucleotide. As
known in the art, a probe serves the purpose to detect an analyte
or amplificate. In the case of the process described above, probes
can be used to detect the amplificates of the target nucleic acids.
For this purpose, probes typically carry labels.
"Labels", often referred to as "reporter groups", are generally
groups that make a nucleic acid, in particular oligonucleotides or
modified oligonucleotides, as well as any nucleic acids bound
thereto distinguishable from the remainder of the sample (nucleic
acids having attached a label can also be termed labeled nucleic
acid binding compounds, labeled probes or just probes). Exemplary
labels are fluorescent labels, which are e.g. fluorescent dyes such
as a fluorescein dye, a rhodamine dye, a cyanine dye, and a
coumarin dye. Exemplary fluorescent dyes are FAM, HEX, JA270,
CAL635, Coumarin343, Quasar705, CyanS00, CY5.5, LC-Red 640, LC-Red
705.
Any primer and/or probe may be chemically modified, i.e. the primer
and/or the probe comprise of a modified nucleotide or a
non-nucleotide compound. The probe or the primer is then a modified
oligonucleotide.
A method of nucleic acid amplification is the Polymerase Chain
Reaction (PCR) which is disclosed, among other references, in U.S.
Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR
typically employs two or more oligonucleotide primers that bind to
a selected nucleic acid template (e.g. DNA or RNA). Primers useful
for nucleic acid analysis include oligonucleotides capable of
acting as a point of initiation of nucleic acid synthesis within
the nucleic acid sequences of the target nucleic acids. A primer
can be purified from a restriction digest by conventional methods,
or it can be produced synthetically. The primer can be
single-stranded for maximum efficiency in amplification, but the
primer can be double-stranded. Double-stranded primers are first
denatured, i.e., treated to separate the strands. One method of
denaturing double stranded nucleic acids is by heating. A
"thermostable polymerase" is a polymerase enzyme that is heat
stable, i.e., it is an enzyme that catalyzes the formation of
primer extension products complementary to a template and does not
irreversibly denature when subjected to the elevated temperatures
for the time necessary to effect denaturation of double-stranded
template nucleic acids. Generally, the synthesis is initiated at
the 3' end of each primer and proceeds in the 5' to 3' direction
along the template strand. Thermostable polymerases have e.g. been
isolated from Thermnus flavus, T. ruber, T. thermophilus, T.
aquaticus, T. lacteus, T. ruben, Bacillus stearothermophilus, and
Methanothermus fervidus. Nonetheless, polymerases that are not
thermostable also can be employed in PCR assays provided the enzyme
is replenished.
If the template nucleic acid is double-stranded, it is necessary to
separate the two strands before it can be used as a template in
PCR. Strand separation can be accomplished by any suitable
denaturing method including physical, chemical or enzymatic means.
One method of separating the nucleic acid strands involves heating
the nucleic acid until it is predominately denatured (e.g., greater
than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating
conditions necessary for denaturing template nucleic acid will
depend, e.g., on the buffer salt concentration and the length and
nucleotide composition of the nucleic acids being denatured, but
typically range from about 90.degree. C. to about 105.degree. C.
for a time depending on features of the reaction such as
temperature and the nucleic acid length. Denaturation is typically
performed for about 5 sec. to 9 min. In order to not expose the
respective polymerase like e.g. the Z05 DNA Polymerase to such high
temperatures for too long and thus risking a loss of functional
enzyme, it can be preferred to use short denaturation steps.
If the double-stranded template nucleic acid is denatured by heat,
the reaction mixture is allowed to cool to a temperature that
promotes annealing of each primer to its target sequence on the
target nucleic acids.
The temperature for annealing can be from about 35.degree. C. to
about 70.degree. C., or about 45.degree. C. to about 65.degree. C.,
or about 50.degree. C. to about 60.degree. C., or about 55.degree.
C. to about 58.degree. C. Annealing times can be from about 10 sec.
to about 1 min. (e.g., about 20 sec. to about 50 sec.; about 30
sec. to about 40 sec.). In this context, it can be advantageous to
use different annealing temperatures in order to increase the
inclusivity of the respective assay. In brief, this means that at
relatively low annealing temperatures, primers may also bind to
targets having single mismatches, so variants of certain sequences
can also be amplified. This can be desirable if e.g. a certain
organism has known or unknown genetic mutations which should also
be detected. On the other hand, relatively high annealing
temperatures bear the advantage of providing higher specificity,
since towards higher temperatures the probability of primer binding
to not exactly matching target sequences continuously decreases. In
order to benefit from both phenomena, in some embodiments of the
invention the process described above comprises of annealing at
different temperatures, for example first at a lower, then at a
higher temperature. If, e.g., a first incubation takes place at
55.degree. C. for about 5 cycles, non-exactly matching target
sequences may be (pre-)amplified. This can be followed e.g. by
about 45 cycles at 58.degree. C., providing for higher specificity
throughout the major part of the experiment. This way, potentially
important genetic mutations are not missed, while the specificity
remains relatively high.
The reaction mixture is then adjusted to a temperature at which the
activity of the polymerase is promoted or optimized, i.e., a
temperature sufficient for extension to occur from the annealed
primer to generate products complementary to the nucleic acid to be
analyzed. The temperature should be sufficient to synthesize an
extension product from each primer that is annealed to a nucleic
acid template, but should not be so high as to denature an
extension product from its complementary template (e.g., the
temperature for extension generally ranges from about 40.degree. C.
to 80.degree. C. (e.g., about 50.degree. C. to about 70.degree. C.;
about 65.degree. C.). Extension times can be from about 10 sec. to
about 5 min., or about 15 sec to 2 min., or about 20 sec. to about
1 min., or about 25 sec. to about 35 sec. The newly synthesized
strands form a double-stranded molecule that can be used in the
succeeding steps of the reaction. The steps of strand separation,
annealing, and elongation can be repeated as often as needed to
produce the desired quantity of amplification products
corresponding to the target nucleic acids. The limiting factors in
the reaction are the amounts of primers, thermostable enzyme, and
nucleoside triphosphates present in the reaction. The cycling steps
(i.e., denaturation, annealing, and extension) can be repeated at
least once. For use in detection, the number of cycling steps will
depend, e.g., on the nature of the sample. If the sample is a
complex mixture of nucleic acids, more cycling steps will be
required to amplify the target sequence sufficient for detection.
Generally, the cycling steps are repeated at least about 20 times,
but may be repeated as many as 40, 60, or even 100 times.
PCR can be carried out in which the steps of annealing and
extension are performed in the same step (one-step PCR) or, as
described above, in separate steps (two-step PCR). Performing
annealing and extension together and thus under the same physical
and chemical conditions, with a suitable enzyme such as, for
example, the Z05 DNA polymerase, bears the advantage of saving the
time for an additional step in each cycle, and also abolishing the
need for an additional temperature adjustment between annealing and
extension. Thus, the one-step PCR reduces the overall complexity of
the respective assay.
In general, shorter times for the overall amplification can be
preferred, as the time-to-result is reduced and leads to a possible
earlier diagnosis.
Other nucleic acid amplification methods to be used comprise the
Ligase Chain Reaction (LCR; Wu D. Y. and Wallace R. B., Genomics 4
(1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991)
189-193); Polymerase Ligase Chain Reaction (Barany F., PCR Methods
and Applic, 1 (1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain
Reaction (EP 0439182 A2); 3SR (Kwoh D. Y. et al., Proc. Natl. Acad.
Sci. USA 86 (1989) 1173-1177; Guatelli J. C., et al., Proc. Natl.
Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808); and NASBA (U.S.
Pat. No. 5,130,238). Further, there are strand displacement
amplification (SDA), transcription mediated amplification (TMA),
and Qb-amplification (for a review see e.g. Whelen A. C. and
Persing D. H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson R.
D. and Myers T. W., Curr. Opin. Biotechnol. 4 (1993) 41-47).
The term "Cp value" or "crossing point" value refers to a value
that allows quantification of input target nucleic acids. The Cp
value can be determined according to the second-derivative maximum
method (Van Luu-The, et al., "Improved real-time RT-PCR method for
high-throughput measurements using second derivative calculation
and double correction," BioTechniques, Vol. 38, No. 2, February
2005, pp. 287-293). In the second derivative method, a Cp
corresponds to the first peak of a second derivative curve. This
peak corresponds to the beginning of a log-linear phase. The second
derivative method calculates a second derivative value of the
real-time fluorescence intensity curve, and only one value is
obtained. The original Cp method is based on a locally defined,
differentiable approximation of the intensity values, e.g., by a
polynomial function. Then the third derivative is computed. The Cp
value is the smallest root of the third derivative. The Cp can also
be determined using the fit point method, in which the Cp is
determined by the intersection of a parallel to the threshold line
in the log-linear region (Van Luu-The, el al., BioTechniques, Vol.
38, No. 2, February 2005, pp. 287-293). The Cp value provided by
the LightCycler instrument offered by Roche is calculated according
to the second-derivative maximum method.
The term "PCR efficiency" refers to an indication of cycle to cycle
amplification efficiency. PCR efficiency is calculated for each
condition using the equation: % PCR
efficiency=(10.sup.(-slope)-1).times.100, wherein the slope was
calculated by linear regression with the log copy number plotted on
the y-axis and Cp plotted on the x-axis. PCR efficiency can be
measured using a perfectly matched or mismatched primer
template.
The term "FRET" or "fluorescent resonance energy transfer" or
"Forster resonance energy transfer" refers to a transfer of energy
between at least two chromophores, a donor chromophore and an
acceptor chromophore (referred to as a quencher). The donor
typically transfers the energy to the acceptor when the donor is
excited by light radiation with a suitable wavelength. The acceptor
typically re-emits the transferred energy in the form of light
radiation with a different wavelength. When the acceptor is a
"dark" quencher, it dissipates the transferred energy in a form
other than light. Whether a particular fluorophore acts as a donor
or an acceptor depends on the properties of the other member of the
FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRA
pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used
dark quenchers include Black Hole Quenchers.TM. (BHQ), (Biosearch
Technologies, Inc., Novato, Calif.), Iowa Black.TM. (Integrated DNA
Tech., Inc., Coralville, Iowa), and BlackBerry.TM. Quencher 650
(BBQ-650) (Berry & Assoc., Dexter, Mich.).
The methods set out above can be based on Fluorescence Resonance
Energy Transfer (FRET) between a donor fluorescent moiety and an
acceptor fluorescent moiety. A representative donor fluorescent
moiety is fluorescein, and representative corresponding acceptor
fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and
Cy5.5. Typically, detection includes exciting the sample at a
wavelength absorbed by the donor fluorescent moiety and visualizing
and/or measuring the wavelength emitted by the corresponding
acceptor fluorescent moiety. In the process according to the
invention, detection can be followed by quantitating the FRET. For
example, detection is performed after each cycling step. For
example, detection is performed in real time. By using commercially
available real-time PCR instrumentation (e.g., LightCycler.TM. or
TaqMan.RTM.), PCR amplification and detection of the amplification
product can be combined in a single closed cuvette with
significantly reduced cycling time. Since detection occurs
concurrently with amplification, the real-time PCR methods obviate
the need for manipulation of the amplification product, and
diminish the risk of cross-contamination between amplification
products. Real-time PCR greatly reduces turn-around time and is an
attractive alternative to conventional PCR techniques in the
clinical laboratory.
The following patent applications describe real-time PCR as used in
the LightCycler.RTM. technology: WO 97/46707, WO 97/46714 and WO
97/46712. The LightCycler.RTM. instrument is a rapid thermal cycler
combined with a microvolume fluorometer utilizing high quality
optics. This rapid thermocycling technique uses thin glass cuvettes
as reaction vessels. Heating and cooling of the reaction chamber
are controlled by alternating heated and ambient air. Due to the
low mass of air and the high ratio of surface area to volume of the
cuvettes, very rapid temperature exchange rates can be achieved
within the thermal chamber.
TaqMan.RTM. technology utilizes a single-stranded hybridization
probe labeled with two chromophor moieties. When a first
fluorescent moiety is excited with light of a suitable wavelength,
the absorbed energy is transferred to a second chromophor according
to the principles of FRET. Typical fluorescent dyes used in this
format are for example, among others, FAM, HEX, CY5, JA270, Cyan
and CY5.5. The second chromophore is generally a quencher molecule.
During the annealing step of the PCR reaction, the labeled
hybridization probe binds to the target nucleic acid (i.e., the
amplification product) and is degraded by the 5' to 3' exonuclease
activity of the Taq or another suitable polymerase as known by the
skilled artisan, such as a Z05 polymerase variant, during the
subsequent elongation phase. As a result, the excited fluorescent
moiety and the quencher moiety become spatially separated from one
another. As a consequence, upon excitation of the first fluorescent
moiety in the absence of the quencher, the fluorescence emission
from the first fluorescent moiety can be detected.
In both detection formats described above, the intensity of the
emitted signal can be correlated with the number of original target
nucleic acid molecules.
As an alternative to FRET, an amplification product can be detected
using a double-stranded DNA binding dye such as a fluorescent DNA
binding dye (e.g., SYBRGREEN 1.RTM., or SYBRGOLD.RTM. (Molecular
Probes)). Upon interaction with the double-stranded nucleic acid,
such fluorescent DNA binding dyes emit a fluorescence signal after
excitation with light at a suitable wavelength. A double-stranded
DNA binding dye such as a nucleic acid intercalating dye also can
be used. When double-stranded DNA binding dyes are used, a melting
curve analysis is usually performed for confirmation of the
presence of the amplification product.
Molecular beacons in conjunction with FRET can also be used to
detect the presence of an amplification product using the real-time
PCR methods of the invention. Molecular beacon technology uses a
hybridization probe labeled with a first fluorescent moiety and a
second fluorescent moiety. The second fluorescent moiety is
generally a quencher, and the fluorescent labels are typically
located at each end of the probe. Molecular beacon technology uses
a probe oligonucleotide having sequences that permit secondary
structure formation (e.g. a hairpin). As a result of secondary
structure formation within the probe, both fluorescent moieties are
in spatial proximity when the probe is in solution. After
hybridization to the amplification products, the secondary
structure of the probe is disrupted and the fluorescent moieties
become separated from one another such that after excitation with
light of a suitable wavelength, the emission of the first
fluorescent moiety can be detected.
Thus, in a method according to the invention is the method
described above using FRET, wherein said probes comprise a nucleic
acid sequence that permits secondary structure formation, wherein
said secondary structure formation results in spatial proximity
between said first and second fluorescent moiety.
Efficient FRET can only take place when the fluorescent moieties
are in local proximity and when the emission spectrum of the donor
fluorescent moiety overlaps with the absorption spectrum of the
acceptor fluorescent moiety.
Thus, in an embodiment, said donor and acceptor fluorescent
moieties are within no more than 5 nucleotides of each other on
said probe. In a further embodiment, said acceptor fluorescent
moiety is a quencher.
As described above, in the TaqMan.RTM. format, during the annealing
step of the PCR reaction, the labeled hybridization probe binds to
the target nucleic acid (i.e., the amplification product) and is
degraded by the 5'- to 3'-exonuclease activity of the Taq or
another suitable polymerase as known by the skilled artisan, such
as a Z05 polymerase variant, during the subsequent elongation
phase. Thus, in an embodiment, in the process described above,
amplification employs a polymerase enzyme having 5'- to
3'-exonuclease activity.
It is further advantageous to carefully select the length of the
amplicon that is yielded as a result of the process described
above. Generally, relatively short amplicons increase the
efficiency of the amplification reaction. Thus, an aspect of the
invention is the process described above, wherein the amplified
fragments comprise up to 450 bases, up to 300 bases, up to 200
bases, or up to 150 bases.
A "sequence" is the primary structure of a nucleic acid, i.e. the
specific arrangement of the single nucleobases of which the
respective nucleic acids consists. It has to be understood that the
term "sequence" does not denote a specific type of nucleic acid
such as RNA or DNA, but applies to both as well as to other types
of nucleic acids such as e.g. peptide nucleic acid (PNA) or others.
Where nucleobases correspond to each other, particularly in the
case of uracil (present in RNA) and thymine (present in DNA), these
bases can be considered equivalent between RNA and DNA sequences,
as known in the pertinent art.
Clinically relevant nucleic acids are often DNA which can be
derived from DNA viruses like e.g. Hepatitis B Virus (HBV),
Cytomegalovirus (CMV) and others, or bacteria like e.g. Chlamydia
trachomatis (CT), Neisseria gonorrhoeae (NG) and others. In such
cases, it can be advantageous to use an internal control nucleic
acid consisting of DNA, in order to reflect the target nucleic
acids properties. The terms "cell", "cell line", and "cell culture"
can be used interchangeably and all such designations include
progeny. Thus, the words "transformants" or "transformed cells"
include the primary transformed cell and cultures derived from that
cell without regard to the number of transfers. All progeny may not
be precisely identical in DNA content, due to deliberate or
inadvertent mutations. Mutant progeny that have the same
functionality as screened for in the originally transformed cell
are included in the definition of transformants. The cells can be
prokaryotic or eukaryotic.
The term "control sequences" refers to DNA sequences necessary for
the expression of an operably linked coding sequence in a
particular host organism. The control sequences that are suitable
for procaryotes, for example, include a promoter, optionally an
operator sequence, a ribosome binding site, positive
retroregulatory elements (see U.S. Pat. No. 4,666,848, incorporated
herein by reference), and possibly other sequences. Eucaryotic
cells are known to utilize promoters, polyadenylation signals, and
enhancers.
The terms "restriction endonucleases" and "restriction enzymes"
refer to enzymes, typically bacterial in origin, which cut
double-stranded DNA at or near a specific nucleotide sequence.
Families of amino acid residues having similar side chains are
defined herein. These families include amino acids with basic side
chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., asparagine, glutamine, serine, threonine, tyrosine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine, tryptophan, cysteine, glycine),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine).
The term "reagent solution" is any solution containing at least one
reagent needed or used for PCR purposes. Most typical ingredients
are polymerase, nucleotides, primers, divalent metal ions (such as
magnesium), salts, pH buffering agents, nucleoside triphosphates
(NTPs) or deoxynucleoside triphosphates (dNTPs), one or more
probes, fluorescent dyes (may be attached to probe), nucleic acid
binding agents, and one or more nucleic acid templates. The reagent
may also be another polymerase reaction additive, which has an
influence on the polymerase reaction or its monitoring.
The term "mastermix" refers to a mixture of all or most of the
ingredients or factors necessary for PCR to occur, and in some
cases, all except for the template and primers which are sample and
amplicon specific. Commercially available mastermixes are usually
concentrated solutions. A mastermix may contain all the reagents
common to multiple samples, but it may also be constructed for one
sample only. Using mastermixes helps to reduce pipetting errors and
variations between samples due to differences between pipetted
volumes.
The term "thermostable polymerase" refers to an enzyme that is
stable to heat, is heat resistant and retains sufficient activity
to effect subsequent primer extension reactions after being
subjected to the elevated temperatures for the time necessary to
denature double-stranded nucleic acids. Heating conditions
necessary for nucleic acid denaturation are well known in the art
and are exemplified in U.S. Pat. Nos. 4,965,188 and 4,889,818,
which are incorporated herein by reference. As used herein, a
thermostable polymerase is suitable for use in a temperature
cycling reaction such as PCR. The examples of thermostable nucleic
acid polymerases include Thermus aquaticus Taq DNA polymerase,
Thermus sp. Z05S polymerase, Thermus flavus polymerase, Thermotoga
maritimna polymerases, such as TMA-25 and TMA-30 polymerases, Tth
DNA polymerase, and the like.
A "polymerase with reverse transcriptase activity" is a nucleic
acid polymerase capable of synthesizing DNA based on an RNA
template. It is also capable of replicating a single or
double-stranded DNA once the RNA has been reverse transcribed into
a single strand cDNA. In an embodiment of the invention, the
polymerase with reverse transcriptase activity is thermostable.
In the amplification of an RNA molecule by a DNA polymerase, the
first extension reaction is reverse transcription using an RNA
template, and a DNA strand is produced. The second extension
reaction, using the DNA template, produces a double-stranded DNA
molecule. Thus, synthesis of a complementary DNA strand from an RNA
template by a DNA polymerase provides the starting material for
amplification.
Thermostable DNA polymerases can be used in a coupled, one-enzyme
reverse transcription/amplification reaction. The term
"homogeneous", in this context, refers to a two-step single
addition reaction for reverse transcription and amplification of an
RNA target. By homogeneous it is meant that following the reverse
transcription (RT) step, there is no need to open the reaction
vessel or otherwise adjust reaction components prior to the
amplification step. In a non-homogeneous RT/PCR reaction, following
reverse transcription and prior to amplification one or more of the
reaction components such as the amplification reagents are e.g.
adjusted, added, or diluted, for which the reaction vessel has to
be opened, or at least its contents have to be manipulated. Both
homogeneous and non-homogeneous embodiments are comprised by the
scope of the invention.
Reverse transcription is an important step in an RT/PCR. It is, for
example, known in the art that RNA templates show a tendency
towards the formation of secondary structures that may hamper
primer binding and/or elongation of the cDNA strand by the
respective reverse transcriptase. Thus, relatively high
temperatures for an RT reaction are advantageous with respect to
efficiency of the transcription. On the other hand, raising the
incubation temperature also implies higher specificity, i.e. the RT
primers will not anneal to sequences that exhibit mismatches to the
expected sequence or sequences. Particularly in the case of
multiple different target RNAs, it can be desirable to also
transcribe and subsequently amplify and detect sequences with
single mismatches, e.g. in the case of the possible presence of
unknown or rare substrains or subspecies of organisms in the fluid
sample.
In order to benefit from both advantages described above, i.e. the
reduction of secondary structures and the reverse transcription of
templates with mismatches, the RT incubation can be carried out at
more than one different temperature.
Therefore, an aspect of the invention is the process described
above, wherein said incubation of the polymerase with reverse
transcriptase activity is carried out at different temperatures
from 30.degree. C. to 75.degree. C., or from 45.degree. C. to
70.degree. C., or from 55.degree. C. to 65.degree. C.
As a further important aspect of reverse transcription, long RT
steps can damage the DNA templates that may be present in the fluid
sample. If the fluid sample contains both RNA and DNA species, it
is thus favorable to keep the duration of the RT steps as short as
possible, but at the same time ensuring the synthesis of sufficient
amounts of cDNA for the subsequent amplification and optional
detection of amplificates.
Thus, an aspect of the invention is the process described above,
wherein the period of time for incubation of the polymerase with
reverse transcriptase activity is up to 30 min., 20 min., 15 min.,
12.5 min., 10 min., 5 min., or 1 min.
A further aspect of the invention is the process described above,
wherein the polymerase with reverse transcriptase activity and
comprising a sequence variation is selected from the group
consisting of a) a CS5 DNA polymerase b) a CS6 DNA polymerase c) a
Thermotoga maritima DNA polymerase d) a Thermus aquaticas DNA
polymerase e) a Thermus thermophilus DNA polymerase f) a Thermus
flavus DNA polymerase g) a Thermus filiformis DNA polymerase h) a
Thermnus sp. sps17 DNA polymerase i) a Thermus sp. Z05 DNA
polymerase j) a Thermotoga neapolitana DNA polymerase k) a
Termosipho africanus DNA polymerase l) a Thermus caldophilus DNA
polymerase
Particularly suitable for these requirements are enzymes carrying a
sequence variation in the polymerase domain that enhances their
reverse transcription efficiency in terms of a faster extension
rate.
Therefore, in the process described above, wherein the polymerase
with reverse transcriptase activity is a polymerase variant with
improved nucleic acid extension rate and/or an improved reverse
transcriptase activity relative to the respective wildtype
polymerase.
In an embodiment, in the process described above, the polymerase
with reverse transcriptase activity is a polymerase variant with
improved reverse transcriptase activity relative to the respective
wildtype polymerase.
Polymerases carrying single sequence variations that render them
particularly useful are disclosed in WO 2008/046612. In particular,
polymerases to be used can be sequence modified DNA polymerases
comprising at least the following motif in the polymerase
domain:
T-G-R-L-S-S-Xb7-Xb8-P-N-L-Q-N; wherein Xb7 is an amino acid
selected from S or T and wherein Xb8 is an amino acid selected from
G, T, R, K, or L, wherein the polymerase comprises 3'-5'
exonuclease activity and has an improved nucleic acid extension
rate and/or an improved reverse transcription efficiency relative
to the wildtype DNA polymerase, wherein in said wildtype DNA
polymerase Xb8 is an amino acid selected from D, E or N.
One example are variants of the thermostable DNA polymerase from
Thermus species Z05 (described e.g. in U.S. Pat. No. 5,455,170),
said sequence variations in the polymerase domain as compared with
the respective wildtype enzyme Z05. An embodiment for the method
according to the invention is a Z05 DNA polymerase variant wherein
the amino acid at position 580 is selected from the group
consisting of G, T, R, K and L.
For reverse transcription using a thermostable polymerase,
Mn.sup.2+ can be the divalent cation and is typically included as a
salt, for example, manganese chloride (MnCl.sub.2), manganese
acetate [Mn(OAc).sub.2], or manganese sulfate (MnSO.sub.4). If
MnCl.sub.2 is included in a reaction containing 50 mM Tricine
buffer, for example, the MnCl.sub.2 is generally present at a
concentration of 0.5-7.0 mM; 2.5-3.5 mM is generally present when
200 .mu.M of each dGTP, dATP, dUTP, and dCTP are utilized.
A "modified" thermostable polymerase refers to a polymerase in
which at least one monomer differs from the reference sequence,
such as a native or wild-type form of the polymerase or another
modified form of the polymerase. Exemplary modifications include
monomer insertions, deletions, and substitutions. Modified
polymerases also include chimeric polymerases that have
identifiable component sequences (e.g., structural or functional
domains, etc.) derived from two or more parents. Also included
within the definition of modified polymerases are those comprising
chemical modifications of the reference sequence. The examples of
modified thermostable polymerases include G46E E678G CS5 DNA
polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G
S671F CS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA
polymerase, a G46E E678G CS6 DNA polymerase, Z05 DNA polymerase,
.DELTA.Z05 polymerase, .DELTA.Z05-Gold polymerase, .DELTA.Z05R
polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase,
E678G TMA-30 polymerase, and the like.
The term "thermoactive polymerase" refers to an enzyme that is
active at the elevated temperatures necessary to ensure specific
priming and primer extension (e.g., 55-80.degree. C.).
The terms "peptide," "polypeptide," and "protein" are used
interchangeably. The terms "nucleic acid" and "polynucleotide" are
used interchangeably. Amino acid sequences are written from amino
terminus to carboxy terminus, unless otherwise indicated.
Single-stranded nucleic acid sequences are written 5' to 3', unless
otherwise indicated. The top strand of a double-stranded nucleic
acid sequence is written 5' to 3', and the bottom strand is written
3' to 5', unless otherwise indicated.
Nucleic acid amplification reagents are typically comprised of
temperature sensitive components, and therefore must often be
stored and shipped at temperatures well below ambient. This is
particularly the case with deoxynucleoside triphosphates or their
ribonucleoside analogs. These reagents are prone to degradation via
loss of consecutive phosphate groups from the terminii, resulting
in the formation of nucleoside diphosphates and monophosphates,
both of which are no longer active as polymerase substrates.
The stability of nucleoside polyphosphates can be improved quite
substantially by esterifying the terminal phosphates. For example,
.gamma.-methyl-dNTP analogs were completely stable under conditions
of heats stress that were sufficient to completely degrade normal
dNTPs. However, esterification of the terminal phosphate can have a
negative effect on the ability of nucleotides to serve as effective
substrates for certain polymerase enzymes.
The present invention describes improved photocleavable ("caged")
nucleoside triphosphates (dNTPs). Installing the thiophenyl
o-nitrophenylpropyl group (SPh-NPP) on the terminal phosphate of
triphosphates stabilizes dNTPs against slow degradation through
hydrolysis. Moreover, the SPh-NPP moiety prevents processing by
enzymes such as polymerases. Irradiation of these compounds with UV
light irreversibly releases free dNTPs and initiates enzymatic
reaction ("Photo-start PCR", see FIG. 1). This strategy suppresses
the formation of non-specific PCR products, which originates from
the residual room temperature activity of the enzyme. In contrast
to hot start PCR, in which the enzyme is activated by heat, the
reaction herein is controlled via light-triggered release of the
dNTP substrate.
Attachment of photocleavable groups has been reported by Ghosn, et
al., Control of DNA Hybridization with Photocleavable Adducts,
Photochem. Pholobiol., 2005, 81:953-959, wherein ester bonds
between the phosphate backbone of DNA oligonucleotides and a
photocleavable group through the intermediacy of
1-(4,5-dimethoxy-2-nitrophenyl)-ethyl ester (DMNPE). More recently,
the thiophenyl extension of the known o-nitrophenyl chromophore was
first introduced by Kretschy el al., Angew. Chem. Int. Ed., 2015,
54Z: 8555-8559. Therein,
thiophenyl-2-(2-nitrophenyl)-propoxycarbonyl (SPh-NPPOC) was used
as a 5'-hydroxyl protecting group on phosphoramidites for
photolithographic microarray synthesis. Photolysis of SPh-NPPOC
releases a nucleoside, a styrene by-product, and also, due to the
carbonate linker, one mole of CO.sub.2. The SPh-NPPOC group
exhibits a 12-fold increase in photo-deprotection efficiency
compared to conventional 2-(2-nitrophenyl)-propoxycarbonyl NPPOC
(FIG. 2).
The stability of the SPh-NPP-dNTPs of the present invention is
especially suitable for use in locations with limited or no
availability of refrigeration. Therefore, the photocleavable dNTPs
of the present invention can be stored much longer and at much
higher temperatures than "conventional" dNTPs.
While the foregoing invention has been described in some detail for
purposes of clarity and understanding, it will be clear to one
skilled in the art from a reading of this disclosure that various
changes in form and detail can be made without departing from the
true scope of the invention. For example, all the compositions and
methods described above can be used in various combinations. All
publications, patents, patent applications, and/or other documents
cited in this application are incorporated by reference in their
entirety for all purposes to the same extent as if each individual
publication, patent, patent application, and/or other document were
individually indicated to be incorporated by reference for all
purposes.
The following examples are given to illustrate embodiments of the
present invention as it is presently preferred to practice. It will
be understood that the examples are illustrative, and that the
invention is not be considered as restricted except as indicated in
the appended claims.
EXAMPLES
Abbreviations: ACN: acetonitrile; CDI: 1,1'-carbonyldiimidazole;
DMF: dimethylformamide: DMSO: dimethylsulfoxide; MeOH: methanol;
TBA: tributylammonium; TEA: trimethylamine; TEAA: triethylammonium
acetate; THF: tetrahydrofuran.
Example 1: Preparation of thiophenyl o-nitrophenylpropyl dATP
(dATP-SPh-NPP)
The synthesis of dATP-SPh-NPP is shown schematically on FIG. 3 and
is briefly described here. All steps were carried out in the dark.
PhSNPP3OH
##STR00005## was dissolved in THF (5 mL, dry) under Ar atmosphere
and cooled to 0-4.degree. C. 220 .mu.L TEA (dry) and 127 &L
POCl.sub.3 were added and the reaction was stirred for 18 h,
quenched with water (8 mL) and stirred for 1.5 h. The aqueous phase
was extracted three times with ethyl acetate. The combined organic
phases were dried over Na.sub.2SO.sub.4. The solvent was removed
under reduced pressure. The residue was dissolved in ACN/H.sub.2O
and lyophilized to give 200 mg of PhSNPP3MP
##STR00006## as a sticky resin, which was directly used for the
next step. 200 mg of raw PhSNPP3MP were dissolved in 6 mL, DMF
(dry), CDI was added and the mixture was stirred for 4 h und dry
conditions. Excess CDI was quenched with MeOH and stirred for 30
min. 52 mg MgCl.sub.2 and then 2.2 eq TBA-ADP solution were added
and the reaction was stirred for 3 days. 0.1 M TEAA buffer (pH 4.1)
was added in a 1:1 ratio. Diaion HP20 resin was added, the mixture
was stirred for 30 min and filtrated. The filtrate was washed with
water and the product was eluted with MeOH. The solvent was then
removed in vacuum, the remaining residue was dissolved in water and
purified by RP18-HPLC. Product fractions were combined and
lyophilized to give PhSNPP3ATP in 51% yield.
Example 2: Stability Studies Using dATP-SPh-NPP
An aqueous mixture was prepared, containing 300 .mu.M dATP-SPh-NPP,
40% DMSO and 1.0 M potassium acetate. For reference another mixture
with 300 .mu.M dATP replacing dATP-SPh-NPP was prepared. 150 .mu.L
of each mixture were incubated at 65.degree. C. in a Thermoshaker.
After 0, 2, 18, 25, and 120 h samples of each solution (20 .mu.L)
were analyzed by UPLC-MS (Ultra performance liquid
chromatography--mass spectrometry). The peak areas at 260 nm were
used to calculate the percentage of starting material and
degradation products.
Result: Under the conditions employed dATP quickly degraded to the
mono- and diphosphates as confirmed by mass spectrometry.
dATP-SPh-NPP is significantly more thermostable than dATP and no
degradation products were observed for dATP-SPh-NPP at 65.degree.
C. for 120 h (FIG. 4).
Example 3: Photoeleavage of dATP-SPh-NPP
Two aqueous mixtures of dATP-SPh-NPP and ATP-DMNPE (300 .mu.M each)
were prepared in sodium phosphate buffer (100 mM, pH 7.0). Both
samples were irradiated simultaneously with light from an UV-LED
lamp (InGaN, Quantum well, .lamda..sub.max=393 nm, 0.18 W/LED) in
open vials without shaking or stirring. After 0, 1, 2, 4, 8, and 16
min. the irradiation was shortly paused for sample removal (20
.mu.L) and subsequent UPLC-MS analysis. The peak areas at 260 nm
were used to calculate the percentage of starting material and
photocleavage products. The molecular identities of the starting
materials and cleavage products were confirmed by mass
spectrometry.
Result: More than >90% dATP was released by irradiating
dATP-SPh-NPP for 5 minutes, and full cleavage was accomplished in 8
minutes (FIG. 5). Photocleavage of dATP-SPh-NPP is significantly
faster than with commercially available ATP-DMNPE (Thermo-Fisher
Scientific).
Example 4: Amplification Reactions Using dATP-SPh-NPP
Real-Time PCR (TaqMan.RTM. PCR) mixtures with a total volume of 50
.mu.L were prepared by combining three components termed PCR master
mixture (20 .mu.L), buffer mixture (20 .mu.L), and dNTP mixture (10
.mu.L). All PCR components were prepared from DEPC water. The PCR
master mixture contained tricine buffer (pH 8.2), potassium
acetate, glycerol, DMSO, detergent, target DNA (400 cp), polymerase
aptamer, probe, primers, SYBR green dye, and polymerase enzyme. The
buffer mixture contained manganese acetate (8.3 mM) and imidazole
(0, 3.0, or 6.0 mM). The dNTP mixture contained dATP (400 .mu.M),
dCTP (400 .mu.M), dGTP (400 .mu.M), and dUTP (800 .mu.M). Another
dNTP mixture contained dATP-SPh-NPP (400 .mu.M) instead of dATP.
The final concentration of DNA target was 400 cp/reaction.
TaqMan.RTM. PCRs of equal composition were positioned on two
separate sections of a 96-well plate. Each section contained PCR
mixtures with regular dNTPs and PCR mixtures containing
dATP-SPh-NPP. Each PCR was prepared as triplicate. One section of
the plate was covered with aluminum foil, whereas the other section
was exposed to light from an UV-LED lamp (InGaN, Quantum well,
.lamda..sub.max=393 nm, 0.18 W/LED) for 8.0 min. at room
temperature. Afterwards the cover was removed and the plate was
subjected to PCR amplification cycles and subsequend DNA melting
with a LightCycler 480 System. The analysis of PCR growth curves
was performed from fluorescence data collected in the Cy5.5
channel, while analysis of melting curves was based on fluorescence
data collected in the FAM channel (465-510 nm).
Result: Caged dATP with SPh-NPP prevents PCR amplification (FIG. 6,
graphs on left column; no UV exposure). Irradiation with UV light
for 8.0 min. and subsequent PCR cycling leads to DNA amplification
(FIG. 6, graphs on right column). The addition of imidazole did not
significantly affect the shapes of the PCR growth curves.
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